Hybrid random bead/chip based microarray

ABSTRACT

A hybrid random bead/chip based microarray includes a plurality of loose optical identification elements (or beads) comprising a substrate having at least one identifiable code disposed thereon. The are randomly placed on a substrate, e.g., tray, plate, slide, or “chip” in an identifiable position (or location) on the slide (not necessarily immobily fixed). For example, the beads are randomly distributed (but oriented) in one dimension along grooves in the slide. Then, each bead code is read and its location determined, i.e., the bead code and location are mapped to its location on the chip. The beads on the chip may then be read/examined (and re-read if desired) by a conventional chip reader. The beads may be functionalized with a desired probe and/or reacted with a desired analyte when in the loose state and/or when on the chip. After reading the chip, the beads may be removed from the chip for further or alternative processing.

CROSS-REFERENCE TO RELATE APPLICATIONS

[0001] This application claims the benefit of US Provisional PatentApplications, Ser. No. 60/441,678, filed Jan. 22, 2003, entitled “HybridRandom Bead/Chip Microarray”, and Ser. No. 60/519,932, filed Nov. 14,2003, entitled, “Diffraction Grating-Based Encoded Microparticles forMultiplexed Experiments”, and is a continuation-in-part of U.S. patentapplication Ser. No. 10/661,234, filed Sep. 12, 2003, entitled“Diffraction Grating-Based Optical Identification Element”; Ser. No.10/661,031, filed Sep. 12, 2003, entitled “Diffraction Grating-BasedEncoded Micro-Particles for Multiplexed Experiments”; and Ser. No.10/661,836, filed Sep. 12, 2003, entitled “Method and Apparatus forAligning Microbeads in order to Interrogate the Same”, of which all theforegoing are incorporated herein by reference in their entirety.

[0002] US Patent Applications Serial Nos. (Atty Docket Nos. CC-0650,CC-0651, CC-0653, and CC-0654), filed Sep. 12, 2003, contain subjectmatter related to that disclosed herein, all of which are incorporatedby reference in their entirety.

TECHNICAL FIELD

[0003] This invention relates to optical identification, and moreparticularly to diffraction grating-based encoded opticalelements/micro-particles for performing multiplexed experiments.

BACKGROUND ART

[0004] A common class of experiments, known as a multiplexed assay ormultiplexed experiment, comprises mixing (or reacting) a labeled targetanalyte or sample (which may have known or unknown properties orsequences) with a set of “probe” or reference substances (which also mayhave known or unknown properties or sequences). Multiplexing allows manyproperties of the target analyte to be probed or evaluatedsimultaneously (i.e., in parallel). For example, in a gene expressionassay, the “target” analyte, usually an unknown sequence of DNA, islabeled with a fluorescent molecule to form the labeled analyte.

[0005] In a known DNA/genomic sequencing assay, each probe consists ofknown DNA sequences of a predetermined length, which are attached to alabeled (or encoded) bead or to a known location or position (or spot)on a substrate.

[0006] When the labeled target analyte is mixed with the probes,segments of the DNA sequence of the labeled target analyte willselectively bind to complementary segments of the DNA sequence of theknown probe. The known probes are then spatially separated and examinedfor fluorescence. The probes that fluoresce indicate that the DNAsequence strands of the target analyte have attached or hybridized tothe complementary DNA of the probe. The DNA sequences in the targetanalyte can then be determined by knowing the complementary DNA (orcDNA) sequence of each known probe to which the labeled target isattached. In addition the level of fluorescence is indicative of howmany target molecules hybridized to the probe molecules for a given beador spot on a substrate.

[0007] Generally, the probes are identified either by spatial locationon a substrate or by attaching the probe to a bead or particle that islabeled (or encoded) to identify the probe, and ultimately the “target”analyte. The first approach separates the probes in a predeterminedgrid, where the probe's identity is linked to its position on the grid.One example of this is a “chip” format, where DNA is attached to a 2-Dsubstrate or microarray, where oligomer DNA sequences are selectivelyattached (either by spotting or grown) onto small sections or spots onthe surface of the substrate in a predetermined spatial order andlocation on a substrate (usually a planar substrate, such as a glassmicroscope slide), such as that sold by Affymetrix and others.

[0008] A second or “bead-based” approach, for identifying the probeallows the probes to mix without any specific spatial position, which isoften called the “random bead assay” approach. In this approach theprobes are attached to a small bead or particle instead of a largersubstrate so they are free to move (usually in a liquid medium). Thisapproach has an advantage in that the analyte reaction can be performedin a liquid/solution by conventional wet-chemistry techniques, whichgives the probes a better opportunity to interact with the analyte.However, this approach requires that each bead or probe be individuallyidentifiable.

[0009] There are many known methods and substrate types that can be usedfor tagging or otherwise uniquely identifying individual beads withattached probes. Known methods include using polystyrene latex spheresthat are colored or fluorescent labeled, such as that sold by Luminexand others. Other methods include using small plastic cans with aconventional bar code applied, or a small container includes a solidsupport material and a radio-frequency tag, such as that sold byPharmaseq and others.

[0010] The beads have the advantage of using liquid or solution basedchemistry and flexibility but current bead technology does have alimited number of identifiable codes and/or are not suitable for harshenvironments/chemicals. Whereas chips typically have the advantage ofhaving higher density (or high multiplexing) capability than beads andcan be read using standard fluorescence scanners, but are not asflexible or economically customizable as beads.

[0011] Therefore, it would be desirable to provide a platform withbenefits of both the bead-based platforms and the chip-based platforms.

SUMMARY OF THE INVENTION

[0012] Objects of the present invention include provision of a platformthat provides benefits of both bead-based platforms and chip-basedplatforms.

[0013] According to the present invention,

[0014] The invention is a significant improvement over chip-based assayplatforms and existing bead-based assay platforms. In particular, thebead assay can be performed with solution or wet chemistry, then whenthe experiment is completed, the beads are placed on a slide, plate, orsubstrate (e.g., a groove plate) which aligns the beads. The beads arethen placed in a “bead mapper”, which reads the codes and maps each beadcode with a unique position on the slide. Once the beads have beenmapped, the slide may be placed in any standard scanner capable ofdetecting the label used for the analyte and its position on the slide.For example, a standard fluorescence reader/scanner used to readchip-based microarrays may be used to read the fluorescence intensity ateach bead location on the slide, similar to reading the fluorescence ofeach spot on the chip. The intensity/location information is thencombined with the code/location information to determine which probesare exhibiting fluorescence, and the intensity thereof.

[0015] The invention may be viewed as a “chip” or “microchip” approachwhere the probes (or beads) are assembled from many individuallyfabricated parts. The beads may be ordered in one dimension along thegrooves, but are randomly distributed (but oriented) along each groove.However, any technique may be used that allows the bead location to beidentified.

[0016] This self-assembled “chip” approach has many advantages overconventional bead based assays. In particular, since the beads are fixedon a chip substrate (e.g., groove plate), they may be examined andre-examined at any time. Also, beads of interest can be easily removedand sorted from the plate/chip after an experiment is performed. Morespecifically, after reading the chip, the beads may be removed from thechip for further and/or alternative processing or experiments. Ifdesired, the chip substrate and/or the beads may be reused in otherexperiments or assays. Further, a fixed plate format is easier to use inexperiments that vary the temperature. Still further, a fixed plateformat allows convenient use of a standard chip reader to examine thebeads. Also, the beads do not need to be examined using a flowcytometer.

[0017] Alternatively, instead of performing the analyte reaction orhybridization reaction before placing the beads on the chip, the beads(or probe particles) can be assembled into the chip format before theanalyte reaction process. In that case, the analyte can be applied tothe chip with the beads disposed thereon, in which case the analytereaction would occur on the chip.

[0018] The microbeads are inexpensive to manufacture and theidentification codes are easy and inexpensive to imprint into themicrobeads. The codes are digitally readable and easily adapted tooptical coding techniques. Thus, the bead mapper optical readout is verysimple and inexpensive to implement. Further, the invention allows forthe use of a standard scanner to the label used for the analyte, whichmay avoid the need to purchase an additional scanner.

[0019] Further, the beads may be oriented in 1-D in grooves (which mayor may not be linear) and are randomly distributed along the grooves.Also, the beads need not be fixed in any way in the grooves other thanby capillary force if desired.

[0020] The code on the bead is not affected by spot imperfections,scratches, cracks or breaks. In addition, splitting or slicing anelement axially produces more elements with the same code; therefore,when a bead is axially split-up, the code is not lost, but insteadreplicated in each piece. Unlike electronic ID elements, the elements ofthe present invention are not affected by nuclear or electromagneticradiation.

[0021] The invention may be used in any assay or multiplexed experiment.The present invention may be used with any known combinatorial chemistryor biochemistry assay process, and are especially adaptable to assayshaving solid phase immobilization. The invention may be used in manyareas such as drug discovery, functionalized substrates, biology,proteomics, combinatorial chemistry, and any assays or multiplexedexperiments. Examples of common assays are SNP (single nucleotidepolymorphism) detection, DNA/genomic sequence analysis, genotyping, geneexpression assays, proteomics assay, peptide assays, antigen/antibodyassays (immunoassay), ligand/receptor assays, DNAanalysis/tracking/sorting/tagging, as well as tagging of molecules,biological particles, cell identification and sorting, matrix supportmaterials, receptor binding assays, scintillation proximity assays,radioactive or non-radioactive proximity assays, and other assays, highthroughput drug/genome screening, and/or massively parallel assayapplications. The analyte can be labeled, detected or identified withany technique capable of being used in an assay with arrays or beads,including but not limited to fluorescent, luminescent, phosphorescent,quantum dot, light scattering colloidal particles, radioactive isotopes,mass spectroscopy, NMR (nuclear magnetic resonance), EPR (electroparamagnetic resonance), ESR (electron spin resonance), IR (infrared),FTIR (Fourier transform infra red), Raman spectroscopy, or othermagenetic, vibrational, electromagnetic, or optical labeling ordetection techniques. Accordingly, the scanner may any scanner capableof measuring or sensing any of the foregoing analyte labels.

[0022] The invention provides uniquely identifiable beads with reactionsupports by active coatings for reaction tracking to perform multiplexedexperiments. The invention may also be used in any chemical and/orbiochemical purification, isolation, or filtering-type process wherebead or bead-like solid supports may be used (e.g., chromatographictechniques, such as affinity column purification). In that case, theabove techniques for labeling, detection or identification may be used.

[0023] The foregoing and other objects, features and advantages of thepresent invention will become more apparent in light of the followingdetailed description of exemplary embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024]FIG. 1 is a side view of an optical identification element, inaccordance with the present invention.

[0025]FIG. 2 is a top level optical schematic for reading a code in anoptical identification element, in accordance with the presentinvention.

[0026]FIG. 3 is a flow chart of a method of using a hybrid randombead/ship based microarray, in accordance with the present invention.

[0027]FIG. 3(a) is a schematic pictorial representation showing a way touse a hybrid random bead/ship based microarray, in accordance with thepresent invention.

[0028]FIG. 4 is a side view of an optical identification element havinga substance attached to the outer surface thereof, in accordance withthe present invention.

[0029]FIG. 5 is a side view of an optical identification element havinga substance attached to the outer surface thereof, in accordance withthe present invention.

[0030]FIG. 6 is a schematic view of a plurality of opticalidentification elements having different identification or codes andcoated with different probe substances disposed in a cell with aplurality of test substances, in accordance with the present invention.

[0031]FIG. 7 is a schematic view of plurality of optical identificationelements, aligned in a plurality of grooves, disposed on a substrate,and a Bead Mapper that scans each optical identification element fordetermining the code and location of each optical identificationelement, in accordance with the present invention.

[0032]FIG. 8 is a side view of an optical identification element afterthe performance of an assay, and a bead detector that determines thecode and fluorescence of the optical identification element, inaccordance with the present invention.

[0033]FIG. 8 is a side view of an optical identification element, and amore detailed view of a Bead Mapper that determines the code andlocation of the optical identification element, in accordance with thepresent invention.

[0034]FIG. 9 is a side view of an optical identification element afterthe performance of an assay, and a more detailed view of aReader/Scanner that reads the fluorescence and location of the opticalidentification element, in accordance with the present invention.

[0035]FIG. 10 is an optical schematic for reading a code in an opticalidentification element, in accordance with the present invention.

[0036]FIG. 11 is an image of a code on a CCD camera from an opticalidentification element, in accordance with the present invention.

[0037]FIG. 12 is a graph showing an digital representation of bits in acode in an optical identification element, in accordance with thepresent invention.

[0038]FIG. 13 illustrations (a)-(c) show images of digital codes on aCCD camera, in accordance with the present invention.

[0039]FIG. 14 illustrations (a)-(d) show graphs of different refractiveindex pitches and a summation graph, in accordance with the presentinvention.

[0040]FIG. 15 is an alternative optical schematic for reading a code inan optical identification element, in accordance with the presentinvention.

[0041]FIG. 16 illustrations (a)-(b) are graphs of reflection andtransmission wavelength spectrum for an optical identification element,in accordance with the present invention.

[0042]FIGS. 17-18 are side views of a thin grating for an opticalidentification element, in accordance with the present invention.

[0043]FIG. 19 is a perspective view showing azimuthal multiplexing of athin grating for an optical identification element, in accordance withthe present invention.

[0044]FIG. 20 is side view of a blazed grating for an opticalidentification element, in accordance with the present invention.

[0045]FIG. 21 is a graph of a plurality of states for each bit in a codefor an optical identification element, in accordance with the presentinvention.

[0046]FIG. 22 is a side view of an optical identification element wherelight is incident on an end face, in accordance with the presentinvention.

[0047]FIGS. 23-24 are side views of an optical identification elementwhere light is incident on an end face, in accordance with the presentinvention.

[0048]FIG. 25, illustrations (a)-(c) are side views of an opticalidentification element having a blazed grating, in accordance with thepresent invention.

[0049]FIG. 26 is a side view of an optical identification element havinga coating, in accordance with the present invention.

[0050]FIG. 27 is a side view of whole and partitioned opticalidentification element, in accordance with the present invention.

[0051]FIG. 28 is a side view of an optical identification element havinga grating across an entire dimension, in accordance with the presentinvention.

[0052]FIG. 29, illustrations (a)-(c), are perspective views ofalternative embodiments for an optical identification element, inaccordance with the present invention.

[0053]FIG. 30, illustrations (a)-(b), are perspective views of anoptical identification element having multiple grating locations, inaccordance with the present invention.

[0054]FIG. 31, is a perspective view of an alternative embodiment for anoptical identification element, in accordance with the presentinvention.

[0055]FIG. 32 is a view an optical identification element having aplurality of gratings located rotationally around the opticalidentification element, in accordance with the present invention.

[0056]FIG. 33 illustrations (a)-(e) show various geometries of anoptical identification element that may have holes therein, inaccordance with the present invention.

[0057]FIG. 34 illustrations (a)-(c) show various geometries of anoptical identification element that may have teeth thereon, inaccordance with the present invention.

[0058]FIG. 35 illustrations (a)-(c) show various geometries of anoptical identification element, in accordance with the presentinvention.

[0059]FIG. 36 is a side view an optical identification element having areflective coating thereon, in accordance with the present invention.

[0060]FIG. 37 illustrations (a)-(b) are side views of an opticalidentification element polarized along an electric or magnetic field, inaccordance with the present invention.

[0061]FIG. 38 is a perspective view of a grooved plate for use with anoptical identification element, in accordance with the presentinvention.

[0062]FIG. 38 is a diagram of the flat grooves and an example of thedimensionality thereof in accordance with the present invention.

[0063]FIG. 40 is a perspective view of a plate with holes for use withan optical identification element, in accordance with the presentinvention.

[0064]FIG. 41 is a perspective view of a grooved plate for use with anoptical identification element, in accordance with the presentinvention.

[0065]FIG. 42 is a diagram of a microbead mapper reading, in accordancewith the present invention.

[0066]FIG. 43 is a diagram of a starting point for handling microbeadsfor readout in a cuvette process in accordance with the invention.

[0067]FIG. 44 is a diagram of showing beads falling into a groove plateor slide, in accordance with the invention.

[0068]FIG. 45 is a diagram of a code readout step for the Bead Mapper,in accordance with the invention.

[0069]FIG. 46 is a diagram of a step of getting beads from a grooveplate back into a tube after being read, in accordance with theinvention.

[0070]FIG. 47 is a diagram of an example of the cuvette or slide showingits mount on a kinematic plate, in accordance with the invention.

[0071]FIG. 48 is a diagram of an alternative embodiment of a cuvetteshowing a port for fluid filling/emptying using a pipette in accordancewith the invention.

[0072]FIG. 49 is a diagram of an alternative embodiment of a cuvetteshowing an alternative port for fluid filling/emptying using a pipettein accordance with the invention.

[0073] FIGS. 50(a), (b) and (c) show embodiments of a disk cytometer inaccordance with the invention.

[0074]FIG. 51(a) show an embodiment of a disk cytometer having radialchannels for spin drying in accordance with the invention.

[0075]FIG. 51(b) show an alternative embodiment of a disk cytometerhaving a mechanical iris for providing a variable aperture for beadaccess to grooves in accordance with the invention.

[0076]FIGS. 52 and 53 are diagrams of bead reads from retro-reflectortrays, in accordance with the present invention.

[0077]FIGS. 54 and 55 are diagrams of bead reads from flatretro-reflector trays, in accordance with the present invention.

[0078]FIGS. 56 and 57 are diagrams of beads read thru V-grooves, inaccordance with the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

[0079] Referring to FIG. 1, a hybrid random bead/chip based microarrayincludes a diffraction grating-based optical identification element 8(or encoded element or coded element) which comprises a known opticalsubstrate 10, having an optical diffraction grating 12 disposed (orwritten, impressed, embedded, imprinted, etched, grown, deposited orotherwise formed) in the volume of or on a surface of a substrate 10.The grating 12 is a periodic or a periodic variation in the effectiverefractive index and/or effective optical absorption of at least aportion of the substrate 10.

[0080] The optical identification element 8 described herein is similarto that described in Copending U.S. patent application Ser. No.10/661,234, filed Sep. 12, 2003, entitled “Diffraction Grating-BasedOptical Identification Element”, which is incorporated herein byreference in its entirety.

[0081] In particular, the substrate 10 has an inner region 20 where thegrating 12 is located. The inner region 20 may be photosensitive toallow the writing or impressing of the grating 12. The substrate 10 hasan outer region 18, which does not have the grating 12 therein.

[0082] The grating 12 is a combination of one or more individual spatialperiodic sinusoidal variations (or components) in the refractive indexthat are collocated at substantially the same location on the substrate10 along the length of the grating region 20, each having a spatialperiod (or pitch) Λ. The resultant combination of these individualpitches is the grating 12, comprising spatial periods (Λ1-Λn) eachrepresenting a bit in the code. Thus, the grating 12 represents a uniqueoptically readable code, made up of bits, where a bit corresponds to aunique pitch Λwithin the grating 12. Accordingly, for a digital binary(0-1) code, the code is determined by which spatial periods (Λ1-Λn)exist (or do not exist) in a given composite grating 12: The code orbits may also be determined by additional parameters (or additionaldegrees of multiplexing), and other numerical bases for the code may beused, as discussed herein and/or in the aforementioned patentapplication.

[0083] The grating 12 may also be referred to herein as a composite orcollocated grating. Also, the grating 12 may be referred to as a“hologram”, as the grating 12 transforms, translates, or filters aninput optical signal to a predetermined desired optical output patternor signal.

[0084] The substrate 10 has an outer diameter D1 and comprises silicaglass (SiO₂) having the appropriate chemical composition to allow thegrating 12 to be disposed therein or thereon. Other materials for theoptical substrate 10 may be used if desired. For example, the substrate10 may be made of any glass, e.g., silica, phosphate glass, borosilicateglass, or other glasses, or made of glass and plastic, or solelyplastic. For high temperature or harsh chemical applications, theoptical substrate 10 made of a glass material is desirable. If aflexible substrate is needed, plastic, rubber or polymer-based substratemay be used. The optical substrate 10 may be any material capable ofhaving the grating 12 disposed in the grating region 20 and that allowslight to pass through it to allow the code to be optically read.

[0085] The optical substrate 10 with the grating 12 has a length L andan outer diameter D1, and the inner region 20 diameter D. The length Lcan range from very small “microbeads” (or microelements,micro-particles, or encoded particles), about 1-1000 microns or smaller,to larger “macroelements” for larger applications (about 1.0-1000 mm orgreater). In addition, the outer dimension D1 can range from small (lessthan 1000 microns) to large (1.0-1000 mm and greater). Other dimensionsand lengths for the substrate 10 and the grating 12 may be used.

[0086] The grating 12 may have a length Lg of about the length L of thesubstrate 10. Alternatively, the length Lg of the grating 12 may beshorter than the total length L of the substrate 10.

[0087] The outer region 18 is made of pure silica (SiO₂) and has arefractive index n2 of about 1.458 (at a wavelength of about 1553 nm),and the inner grating region 20 of the substrate 10 has dopants, such asgermanium and/or boron, to provide a refractive index n1 of about 1.453,which is less than that of outer region 18 by about 0.005. Other indicesof refraction n1,n2 for the grating region 20 and the outer region 18,respectively, may be used, if desired, provided the grating 12 can beimpressed in the desired grating region 20. For example, the gratingregion 20 may have an index of refraction that is larger than that ofthe outer region 18 or grating region 20 may have the same index ofrefraction as the outer region 18 if desired.

[0088] Referring to FIG. 2, an incident light 24 of a wavelength λ,e.g., 532 nm from a known frequency doubled Nd:YAG laser or 632 nm froma known Helium-Neon laser, is incident on the grating 12 in thesubstrate 10. Any other input wavelength λ can be used if desiredprovided λ is within the optical transmission range of the substrate(discussed more herein and/or in the aforementioned patent application).A portion of the input light 24 passes straight through the grating 12,as indicated by a line 25. The remainder of the input light 24 isreflected by the grating 12, as indicated by a line 27 and provided to adetector 29. The output light 27 may be a plurality of beams, eachhaving the same wavelength λ as the input wavelength λ and each having adifferent output angle indicative of the pitches (Λ1-Λn) existing in thegrating 12. Alternatively, the input light 24 may be a plurality ofwavelengths and the output light 27 may have a plurality of wavelengthsindicative of the pitches (Λ1-Λn) existing in the grating 12.Alternatively, the output light may be a combination of wavelengths andoutput angles. The above techniques are discussed in more detail hereinand/or in the aforementioned patent application.

[0089] The detector 29 has the necessary optics, electronics, softwareand/or firmware to perform the functions described herein. Inparticular, the detector reads the optical signal 27 diffracted orreflected from the grating 12 and determines the code based on thepitches present or the optical pattern, as discussed more herein or inthe aforementioned patent application. An output signal indicative ofthe code is provided on a line 31.

[0090] Referring to FIGS. 3-8, and FIG. 3(a), the substrate 10 of theoptical identification element (or microbead) 8 may be functionalized bycoating or attaching a desired probe 76, such as a compound, chemical ormolecule, which is then used in an assay as an attractant for certaincomplimentary compounds, chemicals or molecules, otherwise known as a“target” analyte 52-54 (see FIG. 6). This capability to uniquely encodea large number of microbeads 8 with a corresponding unique probe 76attached thereto enables these functionalized microbeads 72 to be mixedwith unknown “target” analytes 52-54 to perform a multiplexedexperiment.

[0091] Referring to FIGS. 3 and 3(a), a procedure 40 for performing sucha multiplexed assay or experiment using the hybrid random bead/chipbased microarray includes the steps of obtaining (step 41) the microbead8, as described herein, and functionalizing (step 42) the substrate 10of the microbead 8 by coating or depositing or growing it with a probe76 that will react in a predetermined way with “target” analytes 52-54.An assay is then performed (step 43) with a plurality of functionalizedmicrobeads 72 with different identification codes 58 at the same time,e.g., analyte reaction or hybridization, or other multiplexed chemicalreaction or experiment. In step 44, the the microbeads 8 are then placedon a plate, chip or other 2D substrate (as discussed herein), which maybe contained within a housing, chamber or the like (as discussedherein). In step 45, the chip is provided to a Bead Mapper (as discussedherein) which reads the bead codes and bead locations on the chip.

[0092] Next, in step 46, the chip is provided to a Reader/Scanner 824(FIG. 3(a)) where the fluorescence of each of thefunctionalized/hybridized/reacted microbeads 72 is analyzed to determineinformation about the analyte reaction or hybridization for each beadand location. Next a step 47 determines the code 58 of each of the beads72 from the information from the Bead Mapper, thereby determine which“target” analytes 52-54 are present in the solution 60.

[0093] Accordingly, as discussed hereinabove, the assay utilizes thefact that each probe particle (or microbead) is individuallyidentifiable. Once the bead identification code or tag is read, and thespatial position (or location) is known, the self-assembled “chip” canbe inserted into a conventional known chip reader or scanner 824 (FIG.3(a)). The chip reader 824 reads the fluorescent tags on the targetmolecules and determines the spatial location of these tags. Thefluorescent tag location is then used to identify the bead code (andthus probe identification) at that location from the bead mappinginformation to complete the assay or chemical experiment.

[0094] Examples of known chip readers include the following: Axon GenePix Pro 4100 A, GSI/Lumonics/Perkin Elmer Scanner, Alpha Inatech, andothers. Other commercial readers or scanners now known or laterdeveloped may be used provided it can detect the desired analytereaction parameter, e.g., fluorescence, etc., and the it can provide thelocation of same on the substrate.

[0095] Alternatively, the reader/scanner 824 may be similar to theanalyte reaction reading and analysis portions of the microbead readerdevice described in Copending Provisional Patent Applications, Ser. No.60/512,302 (Docket No. CC-0667PR), entitled “Optical Reader forDiffraction Grating Based Encoded Microbeads”, filed Oct. 17, 2003; Ser.No. 60/513,053 (Docket No. CC-0669PR), filed Oct. 21, 2003, “OpticalReader for Diffraction Grating Based Encoded Microbeads”; Ser. No.60/508,038 (Docket No. CC-0577PR), “Optical Reader for DiffractionGrating Based Encoded Microbeads”, filed Oct. 1, 2003, all of which areincorporated herein by reference in their entirety.

[0096] Similarly, the Bead Mapper 20 may be similar to the beadreading/mapping portions of the microbead reader described in CopendingProvisional Patent Applications, Ser. No. 60/512,302 (Docket No.CC-0667PR), entitled “Optical Reader for Diffraction Grating BasedEncoded Microbeads”, filed Oct. 17, 2003; Ser. No. 60/513,053 (DocketNo. CC-0669PR), filed Oct. 21, 2003, “Optical Reader for DiffractionGrating Based Encoded Microbeads”; Ser. No. 60/508,038 (Docket No.CC-0577PR), “Optical Reader for Diffraction Grating Based EncodedMicrobeads”, filed Oct. 1, 2003, all of which are incorporated herein byreference in their entirety.

[0097] In FIGS. 4 and 5, a functionalized microbead 72 is shown, whereinthe substrate 10 of the microbead 8 is coated with a probe 76 and usedin an assay or as an attractant for certain “target” analytes 52-54 (seeFIG. 6). In one embodiment shown in FIG. 4, the microbead 8 is coatedwith a linker molecule or complex 62 as is known in the art. A moleculargroup 64 is attached to the probe 76 to enable the probe to be bonded tothe linker molecule or complex 62, and thus to the microbead 8 to formthe functionalized microbead 72. The probe 76 may include one of anOligonucleitides (oligos), antibodies, peptides, amino acid strings,cDNA, RNA, chemicals, nucleic acid oligomers, polymers, biologicalcells, or proteins. For example, the probe 76 may comprise a singlestrand of DNA (or portion thereof) and the “target” analyte 52-54comprises at least one unknown single strand of DNA, wherein eachdifferent “target” analyte has a different DNA sequence.

[0098] Referring to FIG. 5, in some instances, the probe 76 may beattached directly to the substrate 10 of the microbead 8, or directlysynthesized (or grown) thereon, such as via phosphoramidite chemistry.Examples of surface chemistry for the functionalized microbeads 72include Streptavidin/biotinylated oligos and Aldehyde/amine modifiedoligos. Other chemistry may be used if desired. Some examples ofchemistry are described in Copending Provisional U.S. Patent ApplicationSer. No. 60/519,932, filed Nov. 14, 2003, entitled, “DiffractionGrating-Based Encoded Microparticles for Multiplexed Experiments”, whichis incorporated herein by reference in its entirety. Further, themicrobead may be coated with a blocker of non-specific binding (e.g.,salmon sperm DNA) to prevent bonding of analytes 52-54 (e.g. DNA) to thenon-functionalized surface 66 of the functionalized microbeads 72.

[0099] For example, DNA probe molecules may be directly synthesized onthe beads using standard phosphoramidite chemistry with no postsynthetic purification, and the beads used as the solid support. Theattachment to the bead may be done by preparing the beads using standardlinker chemistry coated on the beads that allows the probe to attach tothe bead. Then, the oligo probe may be grown base-by-base to create theoligo sequence. Alternatively, the entire desired oligo sequence may bepre-fabricated and then attached to the bead after fabrication. In thatcase, the linker chemistry used on the bead would likely be differentand possibly more complex than the linker chemistry used in directsynthesis. Also, the beads may functionalized as discussed hereinbeforeand then placed in a blocker solution of BSA Bovine Serum Albumin (orany other suitable blocker to prevent non-specific binding of the targetmolecule). The beads may then be hybridized by placing the beads in ahybridization solution. Any desirable hybridization solution may beused. One example is: 5× concentration of SSC (Standard Saline Citrate),25% formamide, 0.1% SDS (Sodium Dodecyl Sulfate-soap—used to help thebeads not stick to the walls of tube), a predetermined amount ofcomplementary DNA (cDNA) to the sequence of a given Probe tagged withCy3 fluorescent molecules, and a predetermined amount of complementaryDNA (cDNA) to the sequence of that Probe tagged with Cy5 fluorescentmolecules. Any other hybridization or analyte reaction technique may beused if desired.

[0100] Referring to FIG. 6, an assay is performed by adding a solution60 of different types of “target” analytes 52-54 into a cell orcontainer 70 having a plurality of functionalized microbeads 72-74disposed therein. As discussed in step 46 of FIG. 3, the functionalizedmicrobeads 72-74 placed in the cell 70 have different identificationcodes 58 that correspond to unique probes 76-78 bonded thereto. Forexample, all functionalized microbeads 72 disposed within the cell 70having an identification code of 12345678 is coated with a unique probe76. All functionalized microbeads 73 disposed within the cell 72 havingan identification code of 34128913 is coated with a unique probe 77. Allfunctionalized microbeads 77 disposed within the cell 70 having anidentification code of 11778154 is coated with a unique probe 78.

[0101] The “target” analytes 52-54 within the solution 60 are then mixedwith the functionalized microbeads 72-74. During the mixing of the“target” analytes 52-54 and the functionalized microbeads 72-74, the“target” analytes attach to the complementary probes 76-78, as shown forfunctionalized microbeads 72,73 having codes 12345678 and 34128913.Specifically, as shown in FIG. 6, “target” analytes 53 bonded withprobes 76 of the functionalized microbeads 72 having the code 12345678,and “target” analytes 52 bonded with probes 77 of the functionalizedmicrobeads 73 having the code 34128913. On the other hand, “target”analytes 54 did not bond with any probes, and not “target” analytes52-54 in the solution 60 bonded with probes 78 of the functionalizedmicrobeads 74 having the code 11778154. Consequently, knowing which“target” analytes attach to which probes along with the capability ofidentifying each probe by the encoded microbead, the results of theassay would show that the unknown “target” analytes in the solution 60includes “target” analytes 53, 54, as will be described in furtherdetail.

[0102] For example as discussed hereinbefore, each coded functionalizedmicrobead 72-74 has a unique probe 76-78, respectively bonded thereto,such as a portion of a single strand of DNA. Similarly, the “target”analytes 52-54 comprise a plurality of unknown and unique single strandsof DNA. These “target” analytes 52-54 are also processed with afluorescent, such as dyeing, such that the test molecules illuminate. Aswill be discussed hereinafter, the fluorescence of the “target” analytesprovide the means to identify, which functionalized microbeads 72-74have a “target” analyte attached thereto.

[0103] Once the reaction or combining or hybridization is complete, thefunctionalized (or reacted or hybridized) microbeads 72-74 are rinsedoff with a saline solution to clean off the uncombined “target” analytes52-54.

[0104] Referring to FIG. 7, as discussed herein, the functionalizedmicrobeads 72-74 may be placed on a tray, plate, or substrate (or“chip”) 84 with grooves 82 to allow the microbeads to be aligned in apredetermined direction, such as that described in U.S. PatentApplication Serial No. (Docket No. CC-0648A), filed Sep. 12, 2003, andU.S. Patent Application Serial No. (Docket No. CC-0652), filed Sep. 12,2003, which are both incorporated herein by reference. The grooves 82may have holes (not shown) that provide suction to keep thefunctionalized microbeads in position. Once aligned in the tray 84, thefunctionalized microbeads 52-54 are individually scanned and analyzed bythe bead detector 20.

[0105] Referring to FIGS. 7 and 8, then, each functionalized microbead72-74 is read by a Bead Mapper 20 to determine the identification code58 of each of the functionalized microbeads and the location of eachbead.

[0106] Referring to FIG. 8, more specifically, as discussed herein andin the aforementioned patent applications, the codes in the microbeads 8are detected when illuminated by incident light 24 which produces adiffracted or output light signal 27 to a reader 820, which includes theoptics and electronics necessary to read the codes in each bead 8, asdescribed herein and/or in the aforementioned copending patentapplication. The reader 820 provides a signal on a line 822 indicativeof the code in each of the bead 8 to a known computer 811. The incidentlight 24 may be directed transversely from the side of the tray 84 (orfrom an end or any other angle) with a narrow band (single wavelength)and/or multiple wavelength source, in which case the code is representedby a spatial distribution of light and/or a wavelength spectrum,respectively, as described hereinafter and in the aforementionedcopending patent application. Other illumination, readout techniques,types of gratings, geometries, materials, etc. may be used for themicrobeads 8, as discussed hereinafter and in the aforementioned patentapplication. The computer 811 provides an output signal on a line 813indicative of the bead location and code.

[0107] Referring to FIG. 9, the slide, tray or chip 84 is then placed ina reader or scanner 824 (also see FIG. (3(a)). The reader 824 reads eachfunctionalized microbead 72-74 for fluorescence or other indicator ofthe analyte reaction.

[0108] A light source (not shown) may be provided to luminate themicrobeads 72-74. Once the fluorescent microbeads 72-74 are identifiedand knowing which probe 76-78 (or single strand of DNA) was attached toeach coded, functionalized microbead 72-74, the bead detector 20determines which “target” analytes 52-54 were present in the solution60. As described hereinbefore, the bead detector 20 illuminates thefunctionalized microbeads 72-74 and focuses light 26 reflected by thediffraction grating 12 onto a CCD array or camera 32, whereby the code58 of the functionalized microbead 72-74 is determined. Secondly, thereader 824 includes a fluorescence detector 86 for measuring thefluorescence emanating from “target” analytes 52-54 attached to theprobes 76-78. The fluorescence meter 86 includes a lens 88 and opticalfiber 90 for receiving and providing the fluorescence from the “target”analyte 52-54 to the fluorescence meter.

[0109] Referring to FIG. 9, for assays that use fluorescent moleculemarkers to label or tag chemicals, an optical excitation signal 800 isincident on the microbeads 8 through the tray 84 and a fluorescentoptical output signal 802 emanates from the beads 8 that have thefluorescent molecule attached. The fluorescent optical output signal 802passes through a lens 804, which provides focused light 802 to a knownoptical fluorescence detector 808. Instead of or in addition to the lens802, other imaging optics may be used to provide the desiredcharacteristics of the optical image/signal onto the fluorescencedetector 808. The detector 808 provides an output signal on a line 810indicative of the amount of fluorescence on a given bead 8, which canthen be interpreted to determine what type of chemical is attached tothe bead 10.

[0110] The tray 84 is made of glass or plastic or any material that istransparent to the code reading incident beam 24 and code reading outputlight beams 27 as well as the fluorescent excitation beam 800 and theoutput fluorescent optical signal 802, and is properly suited for thedesired application or experiment, e.g., temperature range, harshchemicals, or other application specific requirements.

[0111] The code signal 822 from the bead code reader 820 and thefluorescent signal 810 from the fluorescence detector are provided to aknown computer 812. The computer reads the code associated with eachbead and determines the chemical probe that was attached thereto from apredetermined table that correlates a predetermined relationship betweenthe bead code and the attached probed. In addition, the computer 812reads the fluorescence associated with each bead and determines thesample or analyte that is attached to the bead from a predetermined datathat correlates a predetermined relationship between the fluorescencetag and the analyte attached thereto. The computer 812 then determinesinformation about the analyte and/or the probe as well as about thebonding of the analyte to the probe, and provides such information on adisplay, printout, storage medium or other interface to an operator,scientist or database for review and/or analysis, as indicated by a line815.

[0112] Generally, the assay of the present invention may be used tocarry out any binding assay or screen involving immobilization of one ofthe binding agents. Such solid-phase assays or screens are well known inthe chemical and biochemical arts. For example, such screening mayinvolve specific binding of cells to a molecule (e.g. an antibody orantigen) immobilized on a microbead in the assay followed by analysis todetect whether or to what extent binding occurs. Alternatively, thebeads may subsequently removed from the groove plate for sorting andanalysis via flow cytometry (see e.g. by Needels et al. (1993). Examplesof biological compounds that may be assayed or screened using the assayof the present invention include, e.g. agonists and antagonists for cellmembrane receptors, toxins, venoms, viral epitopes, hormones, sugars,cofactors, peptides, enzyme substrates, drugs inclusive of opiates andsteroids, proteins including antibodies, monoclonal antibodies, antiserareactive with specific antigenic determinants, nucleic acids, lectins,polysaccharides, cellular membranes and organelles. In addition, thepresent invention may be used in any of a large number of well-knownhybridization assays where nucleic acids are immobilized on a surface ofa substrate, e.g. genotyping, polymorphism detection, gene expressionanalysis, fingerprinting, and other methods of DNA- or RNA-based sampleanalysis or diagnosis.

[0113] Any of the great number of isotopic and non-isotopic labeling anddetection methods well-known in the chemical and biochemical assay artmay be used to detect binding with the present invention. Alternatively,spectroscopic methods well-known in the art may be used to determinedirectly whether a molecule is bound to a surface coating in a desiredconfiguration. Spectroscopic methods include e.g., UV-VIS, NMR,EPR, IR,Raman, mass spectrometry and other methods well-known in the art. Forexample, mass spectrometry also is now widely employed for the analysisof biological macromolecules. The method typically involvesimmobilization of a protein on a surface of substrate where it is thenexposed to a ligand binding interaction. Following ligand binding (ornon-binding) the molecule is desorbed from the surface and into aspectrometer using a laser (see, e.g. Merchant and Weinberger, “Recentadvancements in surface-enhanced laser desorption/ionization-time offlight-mass spectrometry,” Electrophoresis 21: 1164-1177 (2000)). Themicrobeads in the assay of the present invention may be used assubstrates in the mass spectrometry detection methods described above.

[0114] Various aspects of the present invention may be conducted in anautomated or semi-automated manner, generally with the assistance ofwell-known data processing methods. Computer programs and other dataprocessing met hods well known in the art may be used to storeinformation including e.g. microbead identifiers, probe sequenceinformation, sample information, and binding signal intensities. Dataprocessing methods well known in the art may be used to read input datacovering the desired characteristics.

[0115] The invention may be used in many areas such as drug discovery,functionalized substrates, biology, proteomics, combinatorial chemistry,DNA analysis/tracking/sorting/tagging, as well as tagging of molecules,biological particles, matrix support materials, immunoassays, receptorbinding assays, scintillation proximity assays, radioactive ornon-radioactive proximity assays, and other assays, (includingfluorescent, mass spectroscopy), high throughput drug/genome screening,and/or massively parallel assay applications. The invention providesuniquely identifiable beads with reaction supports by active coatingsfor reaction tracking to perform multiplexed experiments.

[0116] Some current techniques used in combinatorial chemistry orbiochemistry are described in U.S. Pat. No. 6,294,327, entitled“Apparatus and Method for Detecting Samples Labeled With Material HavingStrong Light Scattering Properties, Using Reflection Mode Light andDiffuse Scattering”, issued Sept. 23, 2001 to Walton et al.; U.S. Pat.No. 6,242,180, entitled “Computer Aided Visualization and AnalysisSystem for Sequence Evaluation”, issued Jun. 5, 2001, to Chee; U.S. Pat.No. 6,309,823 entitled “Arrays of Nucleic Acid Probes for AnalyzingBiotransformation of Genes and Methods of Using the Same”, Oct. 30,2001, to Cronin et al.; U.S. Pat. No. 6,440,667, entitled “Analysis ofTarget Molecules Using an Encoding System”; U.S. Pat. No. 6,355,432,entitled “Products for Detecting Nucleic Acids”; U.S. Pat. No.6,197,506, entitled “Method of Detecting Nucleic Acids”; U.S. Pat. No.6,309,822, entitled “Method for comparing copy number of nucleic acidsequences”; U.S. Pat. No. 5,547,839, entitled “Sequencing of surfaceimmobilized polymers utilizing micro-fluorescence detection”, U.S. Pat.No. 6,383,754, entitled “Binary Encoded Sequence Tags”, and U.S. Pat.No. 6,383,754, entitled “Fixed Address Analysis of Sequence Tags”, whichare all incorporated herein by reference to the extent needed tounderstand the present invention.

[0117] The invention can be used in combinatorial chemistry, activecoating and functionalized polymers, as well as immunoassays, andhybridization reactions. The invention enables millions of parallelchemical reactions, enable large-scale repeated chemical reactions,increase productivity and reduce time-to-market for drug and othermaterial development industries.

[0118] As discussed hereinbefore, although a fluorescent label isprobably most convenient, other sorts of labels, e.g., radioactive,enzyme linked, optically detectable, or spectroscopic labels may beused. An appropriate detection method applicable to the selectedlabeling method can be selected. Suitable labels includeradionucleotides, enzymes, substrates, cofactors, inhibitors, magneticparticles, heavy metal atoms, and particularly fluorescers,chemiluminescers, and spectroscopic labels. Patents teaching the use ofsuch labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149; and 4,366,241.

[0119] With an appropriate label selected, the detection system bestadapted for high resolution and high sensitivity detection may beselected. As indicated above, an optically detectable system, e.g.,fluorescence or chemilumnescence would be preferred but is not required.Other detection systems may be adapted to the purpose, e.g., electronmicroscopy, scanning electron microscopy (SEM), scanning tunnelingelectron microscopy (STEM), infrared microscopy, atomic force microscopy(AFM), electrical conductance, and image plate transfer.

[0120] Referring to FIG. 10, The reflected light 27, comprises aplurality of beams 26-36 that pass through a lens 37, which providesfocused light beams 46-56, respectively, which are imaged onto a CCDcamera 60. The lens 37 and the camera 60, and any other necessaryelectronics or optics for performing the functions described herein,make up the reader 29. Instead of or in addition to the lens 37, otherimaging optics may be used to provide the desired characteristics of theoptical image/signal onto the camera 60 (e.g., spots, lines, circles,ovals, etc.), depending on the shape of the substrate 10 and inputoptical signals. Also, instead of a CCD camera other devices may be usedto read/capture the output light.

[0121] Referring to FIG. 11, the image on the CCD camera 60 is a seriesof illuminated stripes indicating ones and zeros of a digital pattern orcode of the grating 12 in the element 8. Referring to FIG. 12, lines 68on a graph 70 are indicative of a digitized version of the image of FIG.11 as indicated in spatial periods (Λ1-Λn).

[0122] Each of the individual spatial periods (Λ1-Λn) in the grating 12is slightly different, thus producing an array of N unique diffractionconditions (or diffraction angles) discussed more hereinafter. When theelement 8 is illuminated from the side, in the region of the grating 12,at an appropriate input angle, e.g., about 30 degrees, with a singleinput wavelength λ (monochromatic) source, the diffracted (or reflected)beams 26-36 are generated. Other input angles θi may be used if desired,depending on various design parameters as discussed herein and/or in theaforementioned patent application, and provided that a known diffractionequation (Eq. 1 below) is satisfied:

sin(θ_(i))+sin(θ_(o))=mλ/nΛ  Eq. 1

[0123] where Eq. 1 is diffraction (or reflection or scatter)relationship between input wavelength λ, input incident angle θi, outputincident angle θo, and the spatial period Λ of the grating 12. Further,m is the “order” of the reflection being observed, and n is therefractive index of the substrate 10. The value of m=1 or first orderreflection is acceptable for illustrative purposes. Eq. 1 applies tolight incident on outer surfaces of the substrate 10 which are parallelto the longitudinal axis of the grating (or the k_(B) vector). Becausethe angles θi,θo are defined outside the substrate 10 and because theeffective refractive index of the substrate 10 is substantially a commonvalue, the value of n in Eq. 1 cancels out of this equation.

[0124] Thus, for a given input wavelength λ, grating spacing Λ, andincident angle of the input light θi, the angle θo of the reflectedoutput light may be determined. Solving Eq. 1 for θo and plugging inm=1, gives:

θo=sin⁻¹(λ/Λ−sin(θi))  Eq. 2

[0125] For example, for an input wavelength λ=532 nm, a grating spacingA=0.532 microns (or 532 nm), and an input angle of incidence θi=30degrees, the output angle of reflection will be θo=30 degrees.Alternatively, for an input wavelength λ=632 nm, a grating spacingA=0.532 microns (or 532 nm), and an input angle θi of 30 degrees, theoutput angle of reflection θo will be at 43.47 degrees, or for an inputangle θi=37 degrees, the output angle of reflection will be θo=37degrees. Any input angle that satisfies the design requirementsdiscussed herein and/or in the aforementioned patent application may beused.

[0126] In addition, to have sufficient optical output power and signalto noise ratio, the output light 27 should fall within an acceptableportion of the Bragg envelope (or normalized reflection efficiencyenvelope) curve 200, as indicated by points 204,206, also defined as aBragg envelope angle θB, as also discussed herein and/or in theaforementioned patent application. The curve 200 may be defined as:$\begin{matrix}{{I( {{ki},{ko}} )} \approx {\lbrack{KD}\rbrack^{2}\sin \quad {c^{2}\lbrack \frac{( {{ki} - {ko}} )D}{2} \rbrack}}} & {{Eq}.\quad 3}\end{matrix}$

[0127] where K=2πδn/λ, where, δn is the local refractive indexmodulation amplitude of the grating and λ is the input wavelength,sinc(x)=sin(x)/x, and the vectors k_(i)=2πcos(θ_(i))/λ and k_(o)=2πcos(θ_(o))/λ are the projections of the incident light and the output (orreflected) light, respectively, onto the line 203 normal to the axialdirection of the grating 12 (or the grating vector k_(B)), D is thethickness or depth of the grating 12 as measured along the line 203(normal to the axial direction of the grating 12). Other substrateshapes than a cylinder may be used and will exhibit a similar peakedcharacteristic of the Bragg envelope. We have found that a value for δnof about 104 in the grating region of the substrate is acceptable;however, other values may be used if desired.

[0128] Rewriting Eq. 3 gives the reflection efficiency profile of theBragg envelope as: $\begin{matrix}{{I( {{ki},{ko}} )} \approx {\lbrack \frac{2{\pi \cdot \delta}\quad {n \cdot D}}{\lambda} \rbrack^{2}\lbrack \frac{{Sin}\quad (x)}{x} \rbrack}^{2}} & {{Eq}.\quad 4}\end{matrix}$

[0129] where: x=(ki−ko)D/2=(πD/λ)*(cos θi−cos θo)

[0130] Thus, when the input angle θi is equal to the output (orreflected) angle θ_(o) (i.e., θi=θ_(o)), the reflection efficiency I(Eqs. 3 & 4) is maximized, which is at the center or peak of the Braggenvelope. When θi=θo, the input light angle is referred to as the Braggangle as is known. The efficiency decreases for other input and outputangles (i.e., θi≠θ_(o)), as defined by Eqs. 3 & 4. Thus, for maximumreflection efficiency and thus output light power, for a given gratingpitch Λand input wavelength, the angle θi of the input light 24 shouldbe set so that the angle θo of the reflected output light equals theinput angle θi.

[0131] Also, as the thickness or diameter D of the grating decreases,the width of the sin(x)/x function (and thus the width of the Braggenvelope) increases and, the coefficient to or amplitude of the sinc²(or(sin(x)/x)² function (and thus the efficiency level across the Braggenvelope) also increases, and vice versa. Further, as the wavelength λincreases, the half-width of the Bragg envelope as well as theefficiency level across the Bragg envelope both decrease. Thus, there isa trade-off between the brightness of an individual bit and the numberof bits available under the Bragg envelope. Ideally, δn should be madeas large as possible to maximize the brightness, which allows D to bemade smaller.

[0132] From Eq. 3 and 4, the half-angle of the Bragg envelope θ_(B) isdefined as: $\begin{matrix}{\theta_{B} = \frac{\eta \quad \lambda}{\pi \quad D\quad {\sin ( \theta_{i} )}}} & {{Eq}.\quad 5}\end{matrix}$

[0133] where η is a reflection efficiency factor which is the value forx in the sinc²(x) function where the value of sinc²(x) has decreased toa predetermined value from the maximum amplitude as indicated by points204,206 on the curve 200.

[0134] We have found that the reflection efficiency is acceptable whenη≦1.39. This value for η corresponds to when the amplitude of thereflected beam (i.e., from the sinc²(x) function of Eqs. 3 & 4) hasdecayed to about 50% of its peak value. In particular, when x=1.39=η,sinc²(x)=0.5. However, other values for efficiency thresholds or factorin the Bragg envelope may be used if desired.

[0135] The beams 26-36 are imaged onto the CCD camera 60 to produce thepattern of light and dark regions 120-132 representing a digital (orbinary) code, where light=1 and dark=0 (or vice versa). The digital codemay be generated by selectively creating individual index variations (orindividual gratings) with the desired spatial periods Λ1-Λn. Otherillumination, readout techniques, types of gratings, geometries,materials, etc. may be used as discussed in the aforementioned patentapplication.

[0136] Referring to FIG. 13, illustrations (a)-(c), for the grating 12in a cylindrical substrate 10 having a sample spectral 17 bit code(i.e., 17 different pitches Λ1-Λ17), the corresponding image on the CCD(Charge Coupled Device) camera 60 is shown for a digital pattern of 7bits turned on (10110010001001001); 9 bits turned on of(11000101010100111); all 17 bits turned on of (11111111111111).

[0137] For the images in FIG. 13, the length of the substrate 10 was 450microns, the outer diameter D1 was 65 microns, the inner diameter D was14 microns, δn for the grating 12 was about 104, n1 in portion 20 wasabout 1.458 (at a wavelength of about 1550 nm), n2 in portion 18 wasabout 1.453, the average pitch spacing Λ for the grating 12 was about0.542 microns, and the spacing between pitches ΔΛ was about 0.36% of theadjacent pitches Λ.

[0138] Referring to FIG. 14, illustration (a), the pitch Λof anindividual grating is the axial spatial period of the sinusoidalvariation in the refractive index n1 in the region 20 of the substrate10 along the axial length of the grating 12 as indicated by a curve 90on a graph 91. Referring to FIG. 14, illustration (b), a samplecomposite grating 12 comprises three individual gratings that areco-located on the substrate 10, each individual grating having slightlydifferent pitches, Λ1, Λ2, Λ3, respectively, and the difference (orspacing) ΔΛ between each pitch Λ being about 3.0% of the period of anadjacent pitch Λ as indicated by a series of curves 92 on a graph 94.Referring to FIG. 14, illustration (c), three individual gratings, eachhaving slightly different pitches, Λ1, Λ2, Λ3, respectively, are shown,the difference ΔΛ between each pitch Λ being about 0.3% of the pitch Λofthe adjacent pitch as shown by a series of curves 95 on a graph 97. Theindividual gratings in FIG. 14, illustrations (b) and (c) are shown toall start at 0 for illustration purposes; however, it should beunderstood that, the separate gratings need not all start in phase witheach other. Referring to FIG. 14, illustration (d), the overlapping ofthe individual sinusoidal refractive index variation pitches Λ1-Λn inthe grating region 20 of the substrate 10, produces a combined resultantrefractive index variation in the composite grating 12 shown as a curve96 on a graph 98 representing the combination of the three pitches shownin FIG. 14, illustration (b). Accordingly, the resultant refractiveindex variation in the grating region 20 of the substrate 10 may not besinusoidal and is a combination of the individual pitches Λ (or indexvariation).

[0139] The maximum number of resolvable bits N, which is equal to thenumber of different grating pitches Λ (and hence the number of codes),that can be accurately read (or resolved) using side-illumination andside-reading of the grating 12 in the substrate 10, is determined bynumerous factors, including: the beam width w incident on the substrate(and the corresponding substrate length L and grating length Lg), thethickness or diameter D of the grating 12, the wavelength λ of incidentlight, the beam divergence angle θ_(R), and the width of the Braggenvelope θ_(B) (discussed more in the aforementioned patentapplication), and may be determined by the equation: $\begin{matrix}{N \cong \frac{\eta \quad \beta \quad L}{2\quad D\quad {\sin ( \theta_{i} )}}} & {{Eq}.\quad 6}\end{matrix}$

[0140] Referring to FIG. 15, instead of having the input light 24 at asingle wavelength λ (monochromatic) and reading the bits by the angle θoof the output light, the bits (or grating pitches Λ) may beread/detected by providing a plurality of wavelengths and reading thewavelength spectrum of the reflected output light signal. In this case,there would be one bit per wavelength, and thus, the code is containedin the wavelength information of the reflected output signal.

[0141] In this case, each bit (or Λ) is defined by whether itscorresponding wavelength falls within the Bragg envelope, not by itsangular position within the Bragg envelope 200. As a result, it is notlimited by the number of angles that can fit in the Bragg envelope 200for a given composite grating 12, as in the embodiment discussedhereinbefore. Thus, using multiple wavelengths, the only limitation inthe number of bits N is the maximum number of grating pitches A that canbe superimposed and optically distinguished in wavelength space for theoutput beam.

[0142] Referring to FIGS. 15 and 16, illustration (a), the reflectionwavelength spectrum (λ1-λn) of the reflected output beam 310 willexhibit a series of reflection peaks 695, each appearing at the sameoutput Bragg angle θo. Each wavelength peak 695 (λ1-Λn) corresponds toan associated spatial period (Λ1-Λn), which make up the grating 12.

[0143] One way to measure the bits in wavelength space is to have theinput light angle θi equal to the output light angle θo, which is keptat a constant value, and to provide an input wavelength λ that satisfiesthe diffraction condition (Eq. 1) for each grating pitch Λ. This willmaximize the optical power of the output signal for each pitch Λdetectedin the grating 12.

[0144] Referring to 16, illustration (b), the transmission wavelengthspectrum of the transmitted output beam 330 (which is transmittedstraight through the grating 12) will exhibit a series of notches (ordark spots) 696. Alternatively, instead of detecting the reflectedoutput light 310, the transmitted light 330 may be detected at thedetector/reader 308. It should be understood that the optical signallevels for the reflection peaks 695 and transmission notches 696 willdepend on the “strength” of the grating 12, i.e., the magnitude of theindex variation n in the grating 12.

[0145] In FIG. 15, the bits may be detected by continuously scanning theinput wavelength. A known optical source 300 provides the input lightsignal 24 of a coherent scanned wavelength input light shown as a graph304. The source 300 provides a sync signal on a line 306 to a knownreader 308. The sync signal may be a timed pulse or a voltage rampedsignal, which is indicative of the wavelength being provided as theinput light 24 to the substrate 10 at any given time. The reader 308 maybe a photodiode, CCD camera, or other optical detection device thatdetects when an optical signal is present and provides an output signalon a line 309 indicative of the code in the substrate 10 or of thewavelengths present in the output light, which is directly related tothe code, as discussed herein. The grating 12 reflects the input light24 and provides an output light signal 310 to the reader 308. Thewavelength of the input signal is set such that the reflected outputlight 310 will be substantially in the center 314 of the Bragg envelope200 for the individual grating pitch (or bit) being read.

[0146] Alternatively, the source 300 may provide a continuous broadbandwavelength input signal such as that shown as a graph 316. In that case,the reflected output beam 310 signal is provided to a narrow bandscanning filter 318 which scans across the desired range of wavelengthsand provides a filtered output optical signal 320 to the reader 308. Thefilter 318 provides a sync signal on a line 322 to the reader, which isindicative of which wavelengths are being provided on the output signal320 to the reader and may be similar to the sync signal discussedhereinbefore on the line 306 from the source 300. In this case, thesource 300 does not need to provide a sync signal because the inputoptical signal 24 is continuous. Alternatively, instead of having thescanning filter being located in the path of the output beam 310, thescanning filter may be located in the path of the input beam 24 asindicated by the dashed box 324, which provides the sync signal on aline 323.

[0147] Alternatively, instead of the scanning filters 318,324, thereader 308 may be a known optical spectrometer (such as a known spectrumanalyzer), capable of measuring the wavelength of the output light.

[0148] The desired values for the input wavelengths λ (or wavelengthrange) for the input signal 24 from the source 300 may be determinedfrom the Bragg condition of Eq. 1, for a given grating spacing Λ andequal angles for the input light θi and the angle light θo. Solving Eq.1 for λ and plugging in m=1, gives:

λ=Λ[sin(θo)+sin(θi)]  Eq. 7

[0149] It is also possible to combine the angular-based code detectionwith the wavelength-based code detection, both discussed hereinbefore.In this case, each readout wavelength is associated with a predeterminednumber of bits within the Bragg envelope. Bits (or grating pitches Λ)written for different wavelengths do not show up unless the correctwavelength is used.

[0150] Accordingly, the bits (or grating pitches Λ) can be read usingone wavelength and many angles, many wavelengths and one angle, or manywavelengths and many angles.

[0151] Referring to FIG. 17, the grating 12 may have a thickness ordepth D which is comparable or smaller than the incident beam wavelengthλ. This is known as a “thin” diffraction grating (or the full angleBragg envelope is 180 degrees). In that case, the half-angle Braggenvelope θB is substantially 90 degrees; however, δn must be made largeenough to provide sufficient reflection efficiency, per Eqs. 3 and 4. Inparticular, for a “thin” grating, D*δn≈λ/2, which corresponds to a πphase shift between adjacent minimum and maximum refractive index valuesof the grating 12.

[0152] It should be understood that there is still a trade-off discussedhereinbefore with beam divergence angle OR and the incident beam width(or length L of the substrate), but the accessible angular space istheoretically now 90 degrees. Also, for maximum efficiency, the phaseshift between adjacent minimum and maximum refractive index values ofthe grating 12 should approach a π phase shift; however, other phaseshifts may be used.

[0153] In this case, rather than having the input light 24 coming in atthe conventional Bragg input angle θi, as discussed hereinbefore andindicated by a dashed line 701, the grating 12 is illuminated with theinput light 24 oriented on a line 705 orthogonal to the longitudinalgrating vector 705. The input beam 24 will split into two (or more)beams of equal amplitude, where the exit angle θ_(o) can be determinedfrom Eq. 1 with the input angle θ_(i)=0 (normal to the longitudinal axisof the grating 12).

[0154] In particular, from Eq. 1, for a given grating pitch Λ1, the+/−1^(st) order beams (m=+1 and m=−1), corresponds to output beams700,702, respectively. For the +/−2^(nd) order beams (m=+2 and m=−2),corresponds to output beams 704,706, respectively. The 0^(th) order(undefracted) beam (m=0), corresponds to beam 708 and passes straightthrough the substrate. The output beams 700-708 project spectral spotsor peaks 710-718, respectively, along a common plane, shown from theside by a line 709, which is parallel to the upper surface of thesubstrate 10.

[0155] For example, for a grating pitch Λ=1.0 um, and an inputwavelength λ=400 nm, the exit angles θ_(o) are ˜+/−23.6 degrees (form=+/−1), and +/−53.1 degrees (from m=+/−2), from Eq. 1. It should beunderstood that for certain wavelengths, certain orders (e.g., m=+/−2)may be reflected back toward the input side or otherwise not detectableat the output side of the grating 12.

[0156] Alternatively, one can use only the +/−1^(st) order (m=+/−1)output beams for the code, in which case there would be only 2 peaks todetect, 712, 714. Alternatively, one can also use any one or more pairsfrom any order output beam that is capable of being detected.Alternatively, instead of using a pair of output peaks for a givenorder, an individual peak may be used.

[0157] Referring to FIG. 18, if two pitches Λ1,Λ2 exist in the grating12, two sets of peaks will exist. In particular, for a second gratingpitch Λ2, the +/−1^(st) order beams (m=+1 and m=−1), corresponds tooutput beams 720,722, respectively. For the +/−2^(nd) order beams (m=+2and m=−2), corresponds to output beams 724,726, respectively. The 0^(th)order (un-defracted) beam (m=0), corresponds to beam 718 and passesstraight through the substrate. The output beams 720-726 correspondingto the second pitch Λ2 project spectral spots or peaks 730-736,respectively, which are at a different location than the point 710-716,but along the same common plane, shown from the side by the line 709.

[0158] Thus, for a given pitch Λ (or bit) in a grating, a set ofspectral peaks will appear at a specific location in space. Thus, eachdifferent pitch corresponds to a different elevation or output anglewhich corresponds to a predetermined set of spectral peaks. Accordingly,the presence or absence of a particular peak or set of spectral peaksdefines the code.

[0159] In general, if the angle of the grating 12 is not properlyaligned with respect to the mechanical longitudinal axis of thesubstrate 10, the readout angles may no longer be symmetric, leading topossible difficulties in readout. With a thin grating, the angularsensitivity to the alignment of the longitudinal axis of the substrate10 to the input angle θi of incident radiation is reduced or eliminated.In particular, the input light can be oriented along substantially anyangle θi with respect to the grating 12 without causing output signaldegradation, due the large Bragg angle envelope. Also, if the incidentbeam 24 is normal to the substrate 10, the grating 12 can be oriented atany rotational (or azimuthal) angle without causing output signaldegradation. However, in each of these cases, changing the incidentangle θi will affect the output angle θo of the reflected light in apredetermined predictable way, thereby allowing for accurate output codesignal detection or compensation.

[0160] Referring to FIG. 19, for a thin grating, in addition tomultiplexing in the elevation or output angle based on grating pitch Λ,the bits can also be multiplexed in an azimuthal (or rotational) angleθa of the substrate. In particular, a plurality of gratings750,752,754,756 each having the same pitch Λ are disposed in a surface701 of the substrate 10 and located in the plane of the substratesurface 701. The input light 24 is incident on all the gratings750,752,754,756 simultaneously. Each of the gratings provides outputbeams oriented based on the grating orientation. For example, thegrating 750 provides the output beams 764,762, the grating 752 providesthe output beams 766,768, the grating 754 provides the output beams770,772, and the grating 756 provides the output beams 774,776. Each ofthe output beams provides spectral peaks or spots (similar to thatdiscussed hereinbefore), which are located in a plane 760 that isparallel to the substrate surface plane 701. In this case, a singlegrating pitch Λ can produce many bits depending on the number ofgratings that can be placed at different azimuthal (rotational) angleson the surface of the substrate 10 and the number of output beamspectral peaks that can be spatially and optically resolved/detected.Each bit may be viewed as the presence or absence of a pair of peakslocated at a predetermined location in space in the plane 760. Note thatthis example uses only the m=+/−1^(st) order for each reflected outputbeam. Alternatively, the detection may also use the m=+/−2^(nd) order.In that case, there would be two additional output beams and peaks (notshown) for each grating (as discussed hereinbefore) that may lie in thesame plane as the plane 760 and may be on a concentric circle outsidethe circle 760.

[0161] In addition, the azimuthal multiplexing can be combined with theelevation or output angle multiplexing discussed hereinbefore to providetwo levels of multiplexing. Accordingly, for a thin grating, the numberof bits can be multiplexed based on the number of grating pitches Λand/or geometrically by the orientation of the grating pitches.

[0162] Furthermore, if the input light angle θi is normal to thesubstrate 10, the edges of the substrate 10 no longer scatter light fromthe incident angle into the “code angular space”, as discussed hereinand/or in the aforementioned patent application.

[0163] Also, in the thin grating geometry, a continuous broadbandwavelength source may be used as the optical source if desired.

[0164] Referring to FIG. 20, instead of or in addition to the pitches Λin the grating 12 being oriented normal to the longitudinal axis, thepitches may be created at a angle θg. In that case, when the input light24 is incident normal to the surface 792, will produce a reflectedoutput beam 790 having an angle θo determined by Eq. 1 as adjusted forthe blaze angle θg. This can provide another level of multiplexing bitsin the code.

[0165] Referring to FIG. 21, instead of using an optical binary (0-1)code, an additional level of multiplexing may be provided by having theoptical code use other numerical bases, if intensity levels of each bitare used to indicate code information. This could be achieved by havinga corresponding magnitude (or strength) of the refractive index change(δn) for each grating pitch Λ. Four intensity ranges are shown for eachbit number or pitch Λ, providing for a Base-4 code (where each bitcorresponds to 0, 1, 2, or 3). The lowest intensity level, correspondingto a 0, would exist when this pitch Λis not present in the grating 12.The next intensity level 450 would occur when a first low level δn1exists in the grating that provides an output signal within theintensity range corresponding to a 1. The next intensity level 452 wouldoccur when a second higher level δn2 exists in the grating 12 thatprovides an output signal within the intensity range corresponding to a2. The next intensity level 452, would occur when a third higher levelδn3 exists in the grating 12 that provides an output signal within theintensity range corresponding to a 3.

[0166] Referring to FIG. 22, the input light 24 may be incident on thesubstrate 10 on an end face 600 of the substrate 10. In that case, theinput light 24 will be incident on the grating 12 having a moresignificant component of the light (as compared to side illuminationdiscussed hereinbefore) along the longitudinal grating axis 207 of thegrating (along the grating vector k_(B)), as shown by a line 602. Thelight 602 reflects off the grating 12 as indicated by a line 604 andexits the substrate as output light 608. Accordingly, it should beunderstood by one skilled in the art that the diffraction equationsdiscussed hereinbefore regarding output diffraction angle θo also applyin this case except that the reference axis would now be the gratingaxis 207. Thus, in this case, the input and output light angles θi,θo,would be measured from the grating axis 207 and length Lg of the grating12 would become the thickness or depth D of the grating 12. As a result,a grating 12 that is 400 microns long, would result in the Braggenvelope 200 being narrow. It should be understood that because thevalues of n1 and n2 are close to the same value, the slight anglechanges of the light between the regions 18,20 are not shown herein.

[0167] In the case where incident light 610 is incident along the samedirection as the grating vector (Kb) 207, i.e., θi=0 degrees, theincident light sees the whole length Lg of the grating 12 and thegrating provides a reflected output light angle θo=0 degrees, and theBragg envelope 612 becomes extremely narrow, as the narrowing effectdiscussed above reaches a limit. In that case, the relationship betweena given pitch Λ in the grating 12 and the wavelength of reflection λ isgoverned by a known “Bragg grating” relation:

λ=2n _(eff)Λ  Eq. 8

[0168] where n_(eff) is the effective index of refraction of thesubstrate, λ is the input (and output wavelength) and Λ is the pitch.This relation, as is known, may be derived from Eq. 1 where θi=θo=90degrees.

[0169] In that case, the code information is readable only in thespectral wavelength of the reflected beam, similar to that discussedhereinbefore for wavelength based code reading. Accordingly, the inputsignal in this case may be a scanned wavelength source or a broadbandwavelength source. In addition, as discussed hereinbefore for wavelengthbased code reading, the code information may be obtained in reflectionfrom the reflected beam 614 or in transmission by the transmitted beam616 that passes through the grating 12.

[0170] It should be understood that for shapes of the substrate 10 orelement 8 other than a cylinder, the effect of various different shapeson the propagation of input light through the element 8, substrate 10,and/or grating 12, and the associated reflection angles, can bedetermined using known optical physics including Snell's Law, shownbelow:

n _(in) sin θin=n _(out) sin θout  Eq. 9

[0171] where n_(in) is the refractive index of the first (input) medium,and n_(out) is the refractive index of the second (output) medium, andθin and θout are measured from a line 620 normal to an incident surface622.

[0172] Referring to FIG. 23, if the value of n1 in the grating region 20is greater than the value of n2 in the non-grating region 18, thegrating region 20 of the substrate 10 will act as a known opticalwaveguide for certain wavelengths. In that case, the grating region 20acts as a “core” along which light is guided and the outer region 18acts as a “cladding” which helps confine or guide the light. Also, sucha waveguide will have a known “numerical aperture” (θna) that will allowlight that is within the aperture θna to be directed or guided along thegrating axis 207 and reflected axially off the grating 12 and returnedand guided along the waveguide. In that case, the grating 12 willreflect light having the appropriate wavelengths equal to the pitches Λpresent in the grating 12 back along the region 20 (or core) of thewaveguide, and pass the remaining wavelengths of light as the light 632.Thus, having the grating region 20 act as an optical waveguide forwavelengths reflected by the grating 12 allows incident light that isnot aligned exactly with the grating axis 207 to be guided along andaligned with the grating 12 axis 207 for optimal grating reflection.

[0173] If an optical waveguide is used any standard waveguide may beused, e.g., a standard telecommunication single mode optical fiber (125micron diameter or 80 micron diameter fiber with about a 8-10 microndiameter), or a larger diameter waveguide (greater than 0.5 mmdiameter), such as is describe in U.S. patent application Ser. No.09/455,868, filed Dec. 6, 1999, entitled “Large Diameter Waveguide,Grating”. Further, any type of optical waveguide may be used for theoptical substrate 10, such as, a multi-mode, birefringent, polarizationmaintaining, polarizing, multi-core, multi-cladding, or microsturcturedoptical waveguide, or a flat or planar waveguide (where the waveguide isrectangular shaped), or other waveguides.

[0174] Referring to FIG. 24, if the grating 12 extends across the entiredimension D of the substrate, the substrate 10 does not behave as awaveguide for the incident or reflected light and the incident light 24will be diffracted (or reflected) as indicated by lines 642, and thecodes detected as discussed hereinbefore for the end-incidence conditiondiscussed hereinbefore with FIG. 45, and the remaining light 640 passesstraight through.

[0175] Referring to FIG. 25, illustrations (a)-(c), in illustration (a),for the end illumination condition, if a blazed or angled grating isused, as discussed hereinbefore, the input light 24 is coupled out ofthe substrate 10 at a known angle as shown by a line 650. Referring toFIG. 25, illustration (b), alternatively, the input light 24 may beincident from the side and, if the grating 12 has the appropriate blazeangle, the reflected light will exit from the end face 652 as indicatedby a line 654. Referring to FIG. 25, illustration (c), the grating 12may have a plurality of different pitch angles 660,662, which reflectthe input light 24 to different output angles as indicated by lines 664,666. This provides another level of multiplexing (spatially) additionalcodes, if desired.

[0176] The grating 12 may be impressed in the substrate 10 by anytechnique for writing, impressed, embedded, imprinted, or otherwiseforming a diffraction grating in the volume of or on a surface of asubstrate 10. Examples of some known techniques are described in U.S.Pat. Nos. 4,725,110 and 4,807,950, entitled “Method for ImpressingGratings Within Fiber Optics”, to Glenn et al; and U.S. Pat. No.5,388,173, entitled “Method and Apparatus for Forming Aperiodic Gratingsin Optical Fibers”, to Glenn, respectively, and U.S. Pat. No. 5,367,588,entitled “Method of Fabricating Bragg Gratings Using a Silica GlassPhase Grating Mask and Mask Used by Same”, to Hill, and U.S. Pat. No.3,916,182, entitled “Periodic Dielectric Waveguide Filter”, Dabby et al,and U.S. Pat. No. 3,891,302, entitled “Method of Filtering Modes inOptical Waveguides”, to Dabby et al, which are all incorporated hereinby reference to the extent necessary to understand the presentinvention.

[0177] Alternatively, instead of the grating 12 being impressed withinthe substrate material, the grating 12 may be partially or totallycreated by etching or otherwise altering the outer surface geometry ofthe substrate to create a corrugated or varying surface geometry of thesubstrate, such as is described in U.S. Pat. No. 3,891,302, entitled“Method of Filtering Modes in Optical Waveguides”, to Dabby et al, whichis incorporated herein by reference to the extent necessary tounderstand the present invention, provided the resultant opticalrefractive profile for the desired code is created.

[0178] Further, alternatively, the grating 12 may be made by depositingdielectric layers onto the substrate, similar to the way a known thinfilm filter is created, so as to create the desired resultant opticalrefractive profile for the desired code.

[0179] The substrate 10 (and/or the element 8) may have end-viewcross-sectional shapes other than circular, such as square, rectangular,elliptical, clam-shell, D-shaped, or other shapes, and may haveside-view sectional shapes other than rectangular, such as circular,square, elliptical, clam-shell, D-shaped, or other shapes. Also, 3Dgeometries other than a cylinder may be used, such as a sphere, a cube,a pyramid or any other 3D shape. Alternatively, the substrate 10 mayhave a geometry that is a combination of one or more of the foregoingshapes.

[0180] The shape of the element 8 and the size of the incident beam maybe made to minimize any end scatter off the end face(s) of the element8, as is discussed herein and/or in the aforementioned patentapplication. Accordingly, to minimize such scatter, the incident beam 24may be oval shaped where the narrow portion of the oval is smaller thanthe diameter D1, and the long portion of the oval is smaller than thelength L of the element 8. Alternatively, the shape of the end faces maybe rounded or other shapes or may be coated with an antireflectivecoating.

[0181] It should be understood that the size of any given dimension forthe region 20 of the grating 12 may be less than any correspondingdimension of the substrate 10. For example, if the grating 12 hasdimensions of length Lg, depth Dg, and width Wg, and the substrate 12has different dimensions of length L, depth D, and width W, thedimensions of the grating 12 may be less than that of the substrate 12.Thus, the grating 12, may be embedded within or part of a much largersubstrate 12. Also, the element 8 may be embedded or formed in or on alarger object for identification of the object.

[0182] The dimensions, geometries, materials, and material properties ofthe substrate 10 are selected such that the desired optical and materialproperties are met for a given application. The resolution and range forthe optical codes are scalable by controlling these parameters asdiscussed herein and/or in the aforementioned patent application.

[0183] Referring to FIG. 26, the substrate 10 may have an outer coating799, such as a polymer or other material that may be dissimilar to thematerial of the substrate 10, provided that the coating 799 on at leasta portion of the substrate, allows sufficient light to pass through thesubstrate for adequate optical detection of the code. The coating 799may be on any one or more sides of the substrate 10. Also, the coating799 may be a material that causes the element 8 to float or sink incertain fluids (liquid and/or gas) solutions.

[0184] Also, the substrate 10 may be made of a material that is lessdense than certain fluid (liquids and/or gas) solutions, therebyallowing the elements 8 to float or be buoyant or partially buoyant.Also, the substrate may be made of a porous material, such as controlledpore glass (CPG) or other porous material, which may also reduce thedensity of the element 8 and may make the element 8 buoyant orpartially-buoyant in certain fluids.

[0185] Referring to FIG. 27, the grating 12 is axially spatiallyinvariant. As a result, the substrate 10 with the grating 12 (shown as along substrate 21) may be axially subdivided or cut into many separatesmaller substrates 30-36 and each substrate 30-36 will contain the samecode as the longer substrate 21 had before it was cut. The limit on thesize of the smaller substrates 30-36 is based on design and performancefactors discussed herein and/or in the aforementioned patentapplication.

[0186] Referring to FIG. 28, one purpose of the outer region 18 (orregion without the grating 12) of the substrate 10 is to providemechanical or structural support for the inner grating region 20.Accordingly, the entire substrate 10 may comprise the grating 12, ifdesired. Alternatively, the support portion may be completely orpartially beneath, above, or along one or more sides of the gratingregion 20, such as in a planar geometry, or a D-shaped geometry, orother geometries, as described herein and/or in the aforementionedpatent application. The non-grating portion 18 of the substrate 10 maybe used for other purposes as well, such as optical lensing effects orother effects (discussed herein or in the aforementioned patentapplication). Also, the end faces of the substrate 10 need not beperpendicular to the sides or parallel to each other. However, forapplications where the elements 8 are stacked end-to-end, the packingdensity may be optimized if the end faces are perpendicular to thesides.

[0187] Referring to FIG. 29, illustrations (a)-(c), two or moresubstrates 10,250, each having at least one grating therein, may beattached together to form the element 8, e.g., by an adhesive, fusing orother attachment techniques. In that case, the gratings 12,252 may havethe same or different codes.

[0188] Referring to FIG. 30, illustrations (a) and (b), the substrate 10may have multiple regions 80,90 and one or more of these regions mayhave gratings in them. For example, there may be gratings 12,252side-by-side (illustration (a)), or there may be gratings 82-88, spacedend-to-end (illustration (b)) in the substrate 10.

[0189] Referring to FIG. 31, the length L of the element 8 may beshorter than its diameter D, thus, having a geometry such as a plug,puck, wafer, disc or plate.

[0190] Referring to FIG. 32, to facilitate proper alignment of thegrating axis with the angle θi of the input beam 24, the substrate 10may have a plurality of the gratings 12 having the same codes writtentherein at numerous different angular or rotational (or azimuthal)positions of the substrate 10. In particular, two gratings 550, 552,having axial grating axes 551, 553, respectively may have a commoncentral (or pivot or rotational) point where the two axes 551,553intersect. The angle θi of the incident light 24 is aligned properlywith the grating 550 and is not aligned with the grating 552, such thatoutput light 555 is reflected off the grating 550 and light 557 passesthrough the grating 550 as discussed herein. If the element 8 is rotatedas shown by the arrows 559, the angle θi of incident light 24 willbecome aligned properly with the grating 552 and not aligned with thegrating 550 such that output light 555 is reflected off the grating 552and light 557 passes through the grating 552. When multiple gratings arelocated in this rotational orientation, the bead may be rotated asindicated by a line 559 and there may be many angular positions thatwill provide correct (or optimal) incident input angles θi to thegrating. While this example shows a circular cross-section, thistechnique may be used with any shape cross-section.

[0191] Referring to FIG. 33, illustrations (a), (b), (c), (d), and (e)the substrate 10 may have one or more holes located within the substrate10. In illustration (a), holes 560 may be located at various pointsalong all or a portion of the length of the substrate 10. The holes neednot pass all the way through the substrate 10. Any number, size andspacing for the holes 560 may be used if desired. In illustration (b),holes 572 may be located very close together to form a honeycomb-likearea of all or a portion of the cross-section. In illustration (c), one(or more) inner hole 566 may be located in the center of the substrate10 or anywhere inside of where the grating region(s) 20 are located. Theinner hole 566 may be coated with a reflective coating 573 to reflectlight to facilitate reading of one or more of the gratings 12 and/or toreflect light diffracted off one or more of the gratings 12. Theincident light 24 may reflect off the grating 12 in the region 20 andthen reflect off the surface 573 to provide output light 577.Alternatively, the incident light 24 may reflect off the surface 573,then reflect off the grating 12 and provide the output light 575. Inthat case the grating region 20 may run axially or circumferentially 571around the substrate 10. In illustration (d), the holes 579 may belocated circumferentially around the grating region 20 or transverselyacross the substrate 10. In illustration (e), the grating 12 may belocated circumferentially around the outside of the substrate 10, andthere may be holes 574 inside the substrate 10.

[0192] Referring to FIG. 34, illustrations (a), (b), and (c), thesubstrate 10 may have one or more protruding portions or teeth 570,578,580 extending radially and/or circumferentially from the substrate10. Alternatively, the teeth 570, 578,580 may have any other desiredshape.

[0193] Referring to FIG. 35, illustrations (a), (b), (c) a D-shapedsubstrate, a flat-sided substrate and an eye-shaped (or clam-shell orteardrop shaped) substrate 10, respectively, are shown. Also, thegrating region 20 may have end cross-sectional shapes other thancircular and may have side cross-sectional shapes other thanrectangular, such as any of the geometries described herein for thesubstrate 10. For example, the grating region 20 may have a ovalcross-sectional shape as shown by dashed lines 581, which may beoriented in a desired direction, consistent with the teachings herein.Any other geometries for the substrate 10 or the grating region 20 maybe used if desired, as described herein.

[0194] Referring to FIG. 36, at least a portion of a side of thesubstrate 10 may be coated with a reflective coating to allow incidentlight 510 to be reflected back to the same side from which the incidentlight came, as indicated by reflected light 512.

[0195] Referring to FIG. 37, illustrations (a) and (b), alternatively,the substrate 10 can be electrically and/or magnetically polarized, by adopant or coating, which may be used to ease handling and/or alignmentor orientation of the substrate 10 and/or the grating 12, or used forother purposes. Alternatively, the bead may be coated with conductivematerial, e.g., metal coating on the inside of a holy substrate, ormetallic dopant inside the substrate. In these cases, such materials cancause the substrate 10 to align in an electric or magnetic field.Alternatively, the substrate can be doped with an element or compoundthat fluoresces or glows under appropriate illumination, e.g., a rareearth dopant, such as Erbium, or other rare earth dopant or fluorescentor luminescent molecule. In that case, such fluorescence or luminescencemay aid in locating and/or aligning substrates.

[0196] Referring to FIG. 3(a), instead of the Bead Mapper providing thecode and position information directly to the Reader/scanner 824, it mayprovide this data to an Assay Analysis device 901, which may alsoreceived the bead fluorescence or analyte reaction information andposition from the reader/scanner 824. The assay analyzer can thenprovide the assay results as discussed hereinbefore for thereader/scanner.

[0197] The slide or chip may be a slide within a housing, discussedherein, or merely a slide having gooves, such as shown in FIG. 42, withlittle or no additional mechanical hardware attached thereto or usedthereby, also referred to as an open format.

[0198] Referring to FIG. 42, for an open plate format, meaning there isno top to cover the microbeads 8 and the grooves 205. In this mode, themicrobeads 8 are dispensed onto the plate 200 using, for example, apipette tip or syringe tip, although the scope of the invention is notintended to be limited to the manner of depositing the microbeads on theplate. The microbeads 8 may be then agitated by a sonic transducer (notshown), or manipulated with a mechanical wiper (not shown) or some formof spray nozzle (not shown) to encourage all the microbeads 8 to line upin the grooves 205. It has been observed that substantially all themicrobeads naturally line up in the grooves 205 without the need forencouragement. However, there are always some microbeads, that do notfall naturally into the grooves, and these must either be removed fromthe plate 200 or forced to fall into a groove 205. The open formatapproach has the advantages that grooves plate consists just of theplate and no other complicated features such as walls and a top, andpossibly other chambers or channels to allow fluid flow and bubbleremoval. It also has the advantage that it can easily be made with astandard microscope slide, which is designed to fit all conventionalmicro array readers. However, the open format approach may require themicrobeads to be dried out prior to reading, to avoid the possibility ofnon-uniform or unpredictable optical aberrations caused by the unevenevaporation of the buffer solution.

[0199] Referring to FIGS. 38,39,40,41,52-53,54-57, regarding the groovedslide, plate or chip that the beads may be placed in.

[0200] Referring to FIG. 38, one embodiment of a positioning device 200for aligning the microbeads 8 so the longitudinal axis of the microbeadsis in a fixed orientation relative to the code reading or otherdetection device. The positioning device 200 is shown in the form of atray or plate 200 having grooves 205 for align the microbeads 8 and isused in the process as discussed herein. The geometry grooves may bev-shaped, square or rectangular shaped or any other shape based on thedesign requirements.

[0201] As shown, the microbead elements 8 are placed in the tray 200with grooves 205 to allow the elements 8 to be aligned in apredetermined direction for illumination and reading/detection asdiscussed herein. Alternatively, the grooves 205 may have holes 210 thatprovide suction to keep the elements 8 in position.

[0202] Regarding the formation of the grooves, the grooves in the grooveplate may be made in many different ways, including being formed by SU8photoresistant material, mechanically machining; deep reactive ionetching; or injection molding. One advantage of the injection moldingapproach is that the plate can be manufactured in volume at relativelylow cost, and disposed of after the information about the beads isgathered in the assay process. The groove plate may be made of glass,including fused silica, low fluorescence glass, borosilicate glass.Silicon is used because it is reflective so a reflective coating istypically not needed. Alternative, a mirror coating can be applied tothe plate material to achieve the desired reflectivity.

[0203] Referring to FIGS. 38 and 52, alternatively, the surfaces insidethe grooves 205 may be made of or coated with a reflective material thatreflects the incident light. A light beam is incident onto the substrateand diffracted by the grating 12. In particular, the diffracted beam maybe reflected by a surface 520 of the groove 205 and read from the samedirection as the incident beam 24. Alternatively, referring to FIGS. 38and 53, the incident light beam 24 may be diffracted by the grating 12and pass through the upper surface 529 of the groove and reflected offtwo surfaces 526, 528 which are made or coated with a reflective coatingto redirect the output beam upward as a output light beam 530 which maybe detected as discussed hereinbefore. Also see FIGS. 54-57 for possibleretroreflection and pass-through illumination options.

[0204] Referring to FIG. 39, the scope of the invention is not intendedto be limited to any particular groove shape. For example, FIG. 39 showsa diagram a plate 300 having flat grooves 302 instead of V-shapedgrooves shown in FIG. 38. Some characteristics of the grooves accordingto the present invention are as follows:

[0205] The groove width (w) should be at least as wide as the diameterof the bead (D) but not larger than D+15 μm.

[0206] The thickness of the depth of the groove (T) should be at least0.5 times the diameter of the bead so that it sufficiently traps a beadonce it falls into the groove even when it is subjected to mechanicalagitation. The depth should not exceed 1.5 times the diameter of thebead so as to prevent more than one bead from falling into the samegroove location.

[0207] Groove plates have been made using a thick photoresist called SU8and is available from Microchem. The resist is both chemically inert andmechanically robust once fully cured. The groove walls are formed by theresist material, which is deposited onto a glass or substrate.Advantages of this process include the ability to tailor the depth ofgroove by controlling the thickness of the resist material, andvirtually every other geometric attribute through the design of thephoto mask. Because it is photolithographic process, essentially anyshape profile can be made. For example grooves can be made in simplerows, concentric circles, or spirals. Other features such as discretewells, spots and cross hatches can be made as fiducial marks fortracking and positional registration purposes.

[0208] The scope of the invention is also intended to include thegrooves having a flat bottom as shown in FIG. 39 with outwardly taperedwalls.

[0209] Referring to FIG. 40, an alternative embodiment, whereinalignment may be achieved by using a plate 674 having holes 676 slightlylarger than the elements 8 if the light 24 (FIGS. 2 and 4) is incidentalong the grating axis 207. The incident light indicated as 670 isreflected off the grating and exits through the end as a light 672 andthe remaining light passes through the grating and the plate 674 as aline 678. Alternatively, if a blazed grating is used, incident light 670may be reflected out the side of the plate (or any other desired angle),as indicated by a line 680. Alternatively, input light may be incidentfrom the side of the plate 674 and reflected out the top of the plate474 as indicated by a line 684. The light 672 may be a plurality ofseparate light beams or a single light beam that illuminates the entiretray 674 if desired.

[0210] Referring to FIG. 41, an alternative embodiment, wherein thegroove plate discussed hereinbefore with FIG. 38 may be used for the endillumination/readout condition. In this case, the grating 12 may have ablaze angle such that light incident along the axial grating axis willbe reflected upward, downward, or at a predetermined angle for codedetection. Similarly, the input light may be incident on the grating ina downward, upward, or at a predetermined angle and the grating 12 mayreflect light along the axial grating axis for code detection.

[0211] Referring to FIG. 42, regarding microbead mapper 20 readings,microbeads 8 arranged on a plate 200 having grooves 205. As shown, themicrobeads 8 have different codes (e.g. “41101”, “20502”, “41125”) using16-bit, binary symbology), which may be read or detected using thereader or detector configuration described hereinbefore. The codes inthe beads are used to provide a cross reference to determine which probeis attached to which bead, thus allowing the researcher to correlate thechemical content on each bead with the measured fluorescence signal inthe process discussed herein.

[0212] Consistent with that discussed herein, the grooved plate 200 maybe made of glass or plastic or any material that is transparent to thecode reading incident beam 24 and code reading output light beams 27 aswell as the fluorescent excitation beam 800 and the output fluorescentoptical signal 802, and is properly suited for the desired applicationor experiment, e.g., temperature range, harsh chemicals, or otherapplication specific requirements.

[0213] The code signal 822 from the bead code reader 820 and thefluorescent signal 810 from the fluorescence detector are provided to aknown computer 812. The computer 812 reads the code associated with eachbead and determines the chemical probe that was attached thereto from apredetermined table that correlates a predetermined relationship betweenthe bead code and the attached probed. In addition, the computer 812 andreads the fluorescence associated with each bead and determines thesample or analyte that is attached to the bead from a predeterminedtable that correlates a predetermined relationship between thefluorescence tag and the analyte attached thereto. The computer 812 thendetermines information about the analyte and/or the probe as well asabout the bonding of the analyte to the probe, and provides suchinformation on a display, printout, storage medium or other interface toan operator, scientist or database for review and/or analysis,consistent with shown in step 4 of FIG. 1. The sources 801, 803 the codereader 820, the fluorescence optics 804 and detector 808 and thecomputer 812 may all be part of an assay stick reader 824.

[0214] Alternatively, instead of having the code excitation source 801and the fluorescence excitation source 803, the reader 24 may have onlyone source beam which provides both the reflected optical signal 27 fordetermining the code and the fluorescence signal 802 for reading thetagged analyte attached to the beads 8. In that case the input opticalsignal is a common wavelength that performs both functionssimultaneously, or sequentially, if desired.

[0215] The microbeads 8 may be coated with the desired probe compound,chemical, or molecule prior to being placed in the grooved plate 200.Alternatively, the beads 8 may be coated with the probe after beingplaced in the grooved plate 200. As discussed hereinbefore, the probematerial may be an Oligo, cDNA, polymer, or any other desired probecompound, chemical, cell, or molecule for performing an assay.

[0216] The scope of the invention is not intended to be limited to usingor detecting fluorescent molecule markers during the assay process. Forexample, embodiments of the invention are envisioned using and detectionother types of molecular markers in other types of processes.

[0217] Referring to FIGS. 43-49 show the second mode which is called aclosed format, that consists of not only of a groove plate but also atop and at least three walls to hold the solution and the microbeads ina cuvette-like device generally indicated as 500 shown, for example, inFIG. 43.

[0218] In summary, the closed format approach provides a method foreffectively distributing and aligning microbeads during the readoutprocess, as described below:

[0219] The basic process for handling microbeads with a curvette forreadout consists of the following steps:

[0220] (1) FIG. 43 shows a starting point for handling microbeads for areadout. The microbeads start in a test tube. Typical test-tube volumesare 1.5 ml. The microbeads will generally be in a liquid (usually waterwith a small amount of other buffer chemicals to adjust pH and possiblya small amount [˜0.01%] of detergent.) As shown, a bead tube 502contains the microbeads in a solution, which forms part of the assayprocess described herein.

[0221] (2) FIG. 44 shows the bead tube 502 is coupled to a flange 504 ofthe cuvette 500 is inverted and the beads flow onto the groove plate.The cuvette consists of two round flanges that accept test-tubes, atransparent window, and an opposing groove plate. FIG. 47 shows adrawing of a prototype cuvette. The groove plate outer dimensions can beany size, but typical microscope slide dimensions are convenient(1″×3″). The grooves are mechanically or laser cut lengthwise, and havedimensions that are chosen for the exact size of cylindrical microbead.For instance, for a 125 μm diameter bead, grooves of approximately 150μm wide by 150 μm deep are used. One tube carries the microbeads and asmall amount of carrier fluid. The second tube may be larger and holdmore fluid. The purpose of the second tube is to guarantee a certainfluid level in the next step.

[0222] (3) After the cuvette is inverted and the microbeads flow outonto the groove plate side of the cuvette, the microbeads naturallyalign in the grooves via a small amount of rocking or agitation, whichforms part the assay process disccribed herein.

[0223] (4) FIG. 45 shows the readout step, in which, after the beads areall (or nearly all) aligned in the groove plate, the entire plate ismoved (or the readout laser beam is scanned) in order to read the codesof each beam, which forms part of step 3 of the assay process herein. Ineffect, once the microbeads are in the grooves, the entire cuvette ismoved back and forth across a readout beam. The readout beam istransmitted through the cuvette and contains the code bits encoded onthe scattering angles.

[0224] (5) FIG. 46 shows a final step, in which the cuvette is invertedto its original position and the beads flow back into the original tube502, which forms part of the assay process herein. In other words, afterthe readout process, the cuvette is re-inverted and the microbeads flowback into the original test tube.

[0225]FIG. 47 shows an example of a cuvette generally indicated as 700that is mounted on a kinematic base plate 710. As shown, the cuvette 700has a tube 702 for holding the solution with the beads and a top window704 that is a 1 mm thick glass plate having dimensions of about 1″ by3″. The cuvette also has a bottom plate that is a transparent grooveplate. The location pins 712 and lever arm 714 hold the cuvette 700 inplace on the kinematic plate 710.

[0226] One of the key advantages of using the cuvette device is that thepotential to nearly index match the glass microbeads with a buffersolution thereby reducing the divergence of the laser beam caused by thelensing effect of the microbeads, and minimizing scatter form the grooveplate itself.

[0227] Another advantage involves the potential to prevent microbeadsfrom ever stacking up on top of each other, by limiting the spacebetween the bottom and the top plate to be less than twice the diameterof the microbeads.

[0228] Another advantage is that the cover keeps the fluid fromevaporating.

[0229]FIGS. 48-49 show alternative embodiments of the cuvette shown inFIGS. 43-47. As shown, the microbeads are injected into the cuvette byplacing them near the edge of the opening and allowing the surfacetension, or an induced fluid flow, to pull the microbeads into thecuvette, where, because of the limited height between the floor and theceiling of the cuvette, they are confined to move around in a plane,albeit with all the rotational degrees of freedom unconstrained. Once inthe cuvette the microbeads are quickly and sufficiently constrained bythe grooves as the microbeads fall into them. As in the case of the openformat there is still the finite probability that some number ofmicrobeads will not fall into the grooves and must be coaxed in by someform of agitation (ultrasonic, shaking, rocking, etc.).

[0230] An alternative embodiment of the closed approach, which involvessectioning the closed region into two regions, one where the microbeadsare free to move about in a plane, either in a groove or not, and asecond region where the microbeads are trapped in a groove and can onlymove along the axes of a groove. Trapping the microbeads in a groove isaccomplished by further reducing the height of the chamber to the extentthat the microbeads can no longer hop out of a groove. In thisembodiment, the free region is used to pre-align the microbeads into agroove, facilitating the introduction of microbeads into the trappedsection. By tilting this type of cuvette up gravity can be used to pullthe microbeads along a groove from the free region to the trappedregion. Once in the trapped region the microbeads move to the end of thegroove where they stop. Subsequent microbeads will begin to stack upuntil the groove is completely full of microbeads, which are stackedhead to tail. This has the advantage of packing a large number ofmicrobeads into a small area and prevents the microbeads from everjumping out of the grooves. This approach could also be used to alignthe microbeads prior to injection into some form of flow cytometer, or adispensing apparatus.

[0231]FIG. 50(a) shows an embodiment of a cytometer bead reader having adisk, which may be rotating, generally indicated as 1250, having a diskplatform 1252 with circumferential, concentric, grooves 1254 foraligning microbeads 8. As shown, the rotating disk 1250 has varioussectors for processing the microbeads, including a bead loading zone1256, a bead removal zone 1258 and a readout zone 1260.

[0232]FIG. 50(b) shows an alternative embodiment of a rotating diskgenerally indicated as 1200, having a disk platform 1202 with planargroove plates 1204 a, b, c, d, e, f that are shown with grooves orientedin any one or more different ways. One or more of the planar grooveplates 1204 a, b, c, d, e, f may have an optional channel for fluidrun-off, as shown, and a barrier for preventing the microbeads fromflying off the plate. As shown, the window 1262 for reading the beads isin contact with the fluid containing the beads.

[0233]FIG. 50(c) shows an alternative embodiment of a rotating diskgenerally indicated as 1280, having a disk platform 1282 with radialgrooves 1284 a, 1284 b. The disk platform 1282 has a bead loading zone1286 in the center of the disk. One advantage of this embodiment is thatthe opening of the bead loading zone 1286 will also serve to allow therelease of air bubbles that will naturally collect in the center of thedisk due the reduced density of the fluid, which results from thecentrifugal force pushing the fluid radially outwardly. The rotatingdisk 1280 has tight bead packing due to the centrifugal forces due tothe spinning action of the disk. The rotating disk 1280 has a wedgeshape spacer 1288 that keeps the channel at a constant gap width and awall 1290.

[0234]FIG. 51(a) shows an alternative embodiment of a rotating diskgenerally indicated as 1300 having narrow radial channels 1302 for spindrying so liquid is forced out of the circumferential grooves throughthe radial channels. The plate 1300 may have a mechanical catcher 1320coupled thereto for moving radially outwardly in direction 1320 a ifdesired, for recirculating loose beads.

[0235]FIG. 51(b) show an alternative embodiment of a disk cytometer 1400having a mechanical iris 1402 for providing a variable aperture for beadaccess to grooves in accordance with the invention.

[0236] The dimensions and geometries for any of the embodimentsdescribed herein are merely for illustrative purposes and, as such, anyother dimensions may be used if desired, depending on the application,size, performance, manufacturing requirements, or other factors, in viewof the teachings herein.

[0237] It should be understood that, unless stated otherwise herein, anyof the features, characteristics, alternatives or modificationsdescribed regarding a particular embodiment herein may also be applied,used, or incorporated with any other embodiment described herein. Also,the drawings herein are not drawn to scale.

[0238] Although the invention has been described and illustrated withrespect to exemplary embodiments thereof, the foregoing and variousother additions and omissions may be made therein and thereto withoutdeparting from the spirit and scope of the present invention.

What is claimed is:
 1. An optical identification element attached to achemical, comprising: an optical substrate; at least a portion of saidsubstrate having at least one diffraction grating disposed therein, saidgrating having at least one refractive index pitch superimposed at acommon location; the grating providing an output optical signal whenilluminated by an incident light signal; said optical output signalbeing indicative of a code in said substrate; and the chemical beingattached to said substrate.
 2. The apparatus of claim 1 wherein saidsubstrate is made of a glass material.
 3. The apparatus of claim 1wherein said code comprises a plurality of bits.
 4. The apparatus ofclaim 1 wherein the number of pitches is indicative of the number ofsaid bits in said code.
 5. The apparatus of claim 1 wherein saidsubstrate has a length that is less than about 500 microns.
 6. Theapparatus of claim 1 wherein said substrate has a cylindrical shape. 7.The apparatus of claim 1 wherein said grating is a blazed grating. 8.The apparatus of claim 1 wherein said code comprises a plurality ofbits, each bit having a plurality of states.
 9. The apparatus of claim 1wherein said substrate has a reflective coating disposed thereon. 10.The apparatus of claim 1 wherein said substrate is has a magnetic orelectric charge polarization.
 11. The apparatus of claim 1 wherein saidsubstrate has a grating region where said grating and a non-gratingregion where said grating is not located; and wherein said substrate hasa plurality of grating regions.
 12. The apparatus of claim 1 whereinsaid substrate has geometry having holes therein.
 13. The apparatus ofclaim 1 wherein said substrate is has a geometry having protrudingsections.
 14. The apparatus of claim 1 wherein at least a portion ofsaid substrate is has an end cross sectional geometry selected from thegroup: circular, square, rectangular, elliptical, clam-shell, D-shaped,and polygon
 15. The apparatus of claim 1 wherein at least a portion ofsaid substrate is has a side view geometry selected from the group:circular, square, rectangular, elliptical, clam-shell, D-shaped, andpolygon.
 16. The apparatus of claim 1 wherein at least a portion of saidsubstrate is has a 3-D shape selected from the group: sphere, a cube, apyramid.
 17. The apparatus of claim 1 wherein said code comprises atleast a predetermined number of bits, said number being: 3, 5, 7, 9, 10,12, 14, 16, 18, 20, 24, 28, 30, 40, 50, or
 100. 18. A microparticleattached to a chemical comprising: an optical substrate; at least aportion of said substrate having at least one diffraction gratingdisposed therein, said grating having at least one refractive indexpitch superimposed at a common location; the grating providing an outputoptical signal when illuminated by an incident light signal; saidoptical output signal being indicative of a code in said substrate; andthe chemical being attached to said substrate.
 19. A method ofperforming a multiplexed experiment, comprising: obtaining an opticalsubstrate at least a portion of which having a diffraction grating withone or more refractive index pitches superimposed at a common location;attaching a chemical to said substrate; illuminating said substrate withincident light, said substrate providing an output light signal; andreading said output light signal and detecting a code therefrom.